The investigators propose a multifaceted approach to gain new insights into the mechanisms of imprinting, its relation to DNA methylation, and its effect on the cloning of mammals. Their principal hypothesis is that one can interfere experimentally with the establishment and/or maintenance of the "imprinting marks" of imprinted genes in order to generate animals which are devoid of imprinting. For this they will use the sequential inactivation and reactivation of DNA methyltransferase Dnmt-1 as a tool to remove methylation marks specifically from imprinted genes or, alternatively, they will use nuclear cloning of nuclei from nonimprinted ES cells to create "imprint-free" embryos or animals. Inactivation and reactivation of Dnmt-1 will be accomplished by conditional Dnmt-1 alleles controlled by the Cre and Flp recombinases, respectively. The phenotype of "imprint-free" embryos or mice should provide new insights into the role of imprinting in mammalian development. The most spectacular outcome of the experiment would be the generation of imprint-free but otherwise normal mice, a result which would argue against any intrinsic role of imprinting in mammalian development. Finally, they will use imprint-free embryos or mice as a source for isolating novel imprinted genes and they will study the nature of imprinted X chromosome inactivation. These experiments are also relevant for defining some of the parameters that are important for successful cloning of mammals by nuclear transplantation. Presumably, mammalian cloning requires "epigenetic reprogramming" of the somatic genome. An important goal is, therefore, to characterize the methylation changes imposed on the genome of the donor nucleus following transfer into the oocyte. These experiments will test the hypothesis that errors in the maintenance of imprinting marks increase during the life of the donor and that this is a major cause for the low success rate of mammalian cloning by nuclear transplantation. So far it has been not possible to employ gene targeting approaches in species such as rats because ES cells are available only from mice. The cloning of mammals by nuclear transplantation has opened novel and exciting opportunities to employ established gene targeting approaches in species that will permit the establishment of model systems crucial for efforts to study complex diseases such as cancer. This program attempts to define the biological parameters which limit the nuclear cloning approach.